1. Field of the Invention
The invention relates to a novel system and method for the sequential, directional cloning of multiple DNA sequences into a single vector.
2. Description of the Prior Art
The directional ligation of multiple DNA sequences within vectors is often hindered by the inability to force the orientation of subsequently ligated DNA fragments. This necessitates determination of fragment orientation following each ligation event to select recombinant plasmids with the inserts in the correct orientation (Potter, 1996, Biotechniques, 21:198-200). In addition, when attempting to clone a number of unrelated DNA fragments into a single host, the number of usable restriction sites declines rapidly, due to the presence of the sites in the insert DNA(s). Although it is sometimes possible to insert multiple genes into a single vector using a combination of available multi-cloning site (MCS) restriction sites (Jach et al., 1995, Plant Journal, 8:97-109; and Yamano et al., 1994, Biosci. Biotechnol. Biochem., 58:1112-1114), the process is often impractical. Moreover, the process is even more unreliable when attempting to directionally clone more than two genes into the vector.